The present invention relates to a method of and apparatus for separating proteins, in particular proteins from human plasma, from residues of human plasma cryoproteins or residues of cell cultures which proteins are adsorbed by ion-exchange gels, especially anion-exchanging gels like diethylaminoethyl (DEAE) gels. In addition, the present invention refers to pharmaceutical compositions containing such fractions of proteins.
The adsorption of a number of plasma proteins by gels carrying charged groups at their surface and added to blood plasma is known. The surface retention of proteins by these gels is dependent on the functional group of the respectively used gel i.e. whether the functional group is positive or negative and thus adsorbs anions or cations and on the dissociation constant of the charged group, and is further dependent on the prevailing conditions during mixing of the gel with the blood plasma, primarily on the ionic concentration, the kind of dissolved ions, the pH, and the temperature.
When adding to the blood plasma gels having as functional group an anion-exchanging effect as e.g. DEAE-gels, plasma proteins are adsorbed by the gel which are known as "factors of the prothrombin complex". The protein-loaded gel can then simply be separated from the residual plasma. Since the bonding strength between the plasma proteins and the ion-exchanging gels decreases with increasing ionic concentration of the surrounding solution, the proteins can be desorbed from the gel in solutions of high ionic strength. The German patent DE-PS 2 715 832, East German patents DD 148,297 and DD 141,261 and European patent EP 0041173 disclose methods by which the factors of the prothrombin complex are adsorbed from the blood plasma by DEAE-gels and together desorbed without separation into individual components of the prothrombin complex through washing of the gels with solutions of high ionic strength.
It is also known that the prothrombin complex contains various proteins as e.g. the blood clotting factors F II, F VII, FIX F X, as well as blood clotting inhibitors like protein C and protein S. Each component of the prothrombin complex has different biological effects, especially when considering the blood clotting factors and the blood clotting inhibitors which are completely antagonistic to each other.
The medical literature describes genetical diseases which manifest themselves by the incapability of a patient to synthesize one component of the prothrombin complex so that the blood plasma of such a patient lacks this component. A deficiency of one of the clotting factors leads to hemophilia e.g. a deficiency of factor IX results in hemophilia B, while a deficiency of one of both blood clotting inhibitors leads to thrombosis in the patient. The therapy of these completely different diseases comprises, however, the use of an identical plasma derivative that is prothrombin complex to compensate for the deficiency. Evidently, the simultaneous administration of the deficient clotting factor with blood clotting inhibitors may increase the tendency for bleeding in hemophiliacs while in patients suffering of thrombosis, the simultaneous administration of clotting factors in addition to the required inhibitors may further increase the risk of thrombosis.
It is certainly preferable to treat a patient with a deficiency of one clotting factor with a preparation containing only the deficient clotting factor and otherwise being free of inhibitors, and to treat a patient who is deficient of one of the inhibitors with a preparation containing only the concerned inhibitor without any clotting factors.
A method for essentially separating the components of the prothrombin complex is e.g. disclosed by S. P. Bajaj et al. in the publication Preparative Biochemistry, 13 (3), 191-214, 1983. The method comprises a sequence of steps: After saturation of the plasma with ammonium sulphate to 33%, the precipitated proteins are hurled off and the supernatant residue is enriched with ammonium sulphate to 66% or 70% saturation. The precipitated proteins are hurled off and redissolved in saline solution. Barium salt (BaCl or BaSO.sub.4) is added and the precipitated proteins are hurled off and redissolved in saline solution. The precipitation with barium salt may also be carried out prior to the differential precipitation with ammonium sulphate. Subsequently, the proteins are subjected to adsorption on a DEAE-gel column and fractional desorption by gradient elution with a gradient of increasing ionic strength in the elution buffer. After this gel chromatography, the components of the prothrombin complex are, however, not yet entirely separated so that a further step is proposed which comprises a preparative acrylamide electrophoresis.
The above described fractionation method is only suitable to prepare the factors of the prothrombin complex on a laboratory scale for biochemical in vitro tests or for immunization of animals for the production of antisera. According to the prior art, the acrylamide electrophoresis works only with low amounts of protein like less than 5,000 plasma units of e.g. factor IX. Moreover, apart from the electrophoresis, the remaining steps of this method are very time consuming and require the use of protease inhibitors like benzamidine in order to reduce the enzymatic denaturation of the proteins during this lengthy method. As is well documented, these protease inhibitors are of considerable toxicity so that their application in protein preparations for humans is not possible. Further, the use of barium salt for precipitation is undesirable as these compounds are toxic as well.